Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21251
標題: 十字花科蔬菜黑腐病菌Xanthomonas campestris pv. campestris致病基因orfF及其基因產物之定性
Characterization of a virulent gene of Xanthomonas campestris pv. campestris, orfF, and its gene product
作者: 李培增
Lee, Pei-Tseng
關鍵字: Xanthomonas campestris pv. campestris
黃原菌
black root
黑腐病
出版社: 分子生物學研究所
摘要: Xanthomonas campestris pv. campestris (Xcc) 能引起十字花科植物 (crucifers) 的黑腐病 (black rot)。本研究為探討其一致病品系Xcc11的orf F基因及其產物與病原性的關係。首先將一段攜帶有orf F基因的Xcc genomic DNA 片段於orf F基因中插入一段Kmr 基因,再利用一自殺質體將此orf F::Kmr片段送入Xcc11與另一致病品系Xcc17中進行DNA重組。先選取Kmr突變株,再以南方分析確定為orf F::Kmr突變株。以蕪菁幼苗進行病原性測試時,發現Xcc17的orf F::Kmr突變株致病力減弱,而Xcc11的orf F::Kmr突變株則完全喪失致病力。 利用大腸桿菌T7 promoter/ T7 RNA polymerase系統及35S Methionine,證明攜帶有orf F基因的Xcc11 genomic DNA 片段在大腸桿菌中可表現一與Orf F蛋白預測大小相同 (12.4 kDa) 的蛋白產物。進一步以蛋白質表現載體pET21b及親合性層析管柱獲得Orf F-His蛋白溶液。以HPLC分析此Orf F-His蛋白溶液,發現有兩種形式的Orf F-His蛋白存在,分子量皆為約13 kDa。將此Orf F-His蛋白溶液做為抗原,製備抗體,再以此抗體對Xcc11與Xcc17進行西方雜配分析,結果顯示兩品系Xcc菌的菌體蛋白及培養液蛋白中皆可偵測到雜配訊號。此結果顯示Orf F蛋白為一分泌蛋白。 由於Orf F蛋白在第28~30位置的氨基酸具有疑似nuclear localization signal (NLS) 的序列,因此將orf F 基因接到GUS報導基因並選殖入植物表現質體中,利用粒子傳遞系統 (biolistic system) 將質體送入洋蔥上表皮細胞並進行染色,發現Orf F-GUS融合蛋白會進入洋蔥細胞的細胞核中。但若將Orf F的NLS序列進行deletion或mutation後,進行相同的實驗,則發現deletion或mutation的Orf F-GUS融合蛋白就無法進入洋蔥細胞的細胞核中。利用yeast one-hybrid system發現Orf F蛋白在酵母菌中並不具有活化轉錄作用的能力。
Xanthomonas campestris pv. campestris (Xcc) is the causing agent of black rot of crucifies. The purpose of this study is to find out the role which orf F gene plays in pathogenicity. Insertion of a Kmr DNA fragment in the orf F gene was first made with an orf F-containing Xcc genomic DNA fragment, and the fragment was introduced into two strains of Xcc via a suicide vector. The orf F::Kmr recombinants were selected and confirmed by Southern hybridization. The orf F::Kmr mutants of one Xcc strain showed reduced pathogenicity toward turnip plants, whereas those of the other Xcc strain completely lost the pathogenicity. By using the T7 RNA polymerase/T7 promoter system and 35S Methionine, it was found that the orf F-containing Xcc DNA fragment was capable of expressing a protein with expected size of Orf F. With the protein expression plasmid pET21b, an Orf F-His protein was generated in vivo and purified through affinity chromatography. However, further HPLC analysis indicated that the protein solution contained two forms of Orf F-His with same molecular weight. The Orf F-His protein solution was used to prepare polyclonal antibody which was then to probe the total proteins of two Xcc strains and their cultural filtrates. Both strains demostrated hybridization signals with their cellular proteins and the cultrual filtrates, indicating that Orf F is a secretion protein. The orf F gene was cloned into a plant expression vector with GUS reporter gene and delivered into onion epidermal cells via particle biolistic system. It was shown that the Orf F-GUS protein was localized in the nucleus of the onion dermal cells. The 28th to 30th residues of the predictive Orf F protein sequence are lysine residues and likely constitue of a nuclear localization signal. Deletion or mutation of the three residues abolished the nuclear localization capability of Orf F. Further yeast one-hybrid study indicated that Orf F did not have transcriptional activation activity in yeast.
URI: http://hdl.handle.net/11455/21251
Appears in Collections:分子生物學研究所

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